Screening method

Abstract

The invention concerns a method for screening a compound which is a candidate modulator of signal response comprising the steps of; (a) incubating the compound with the PH domain of a signaling molecule which is capable of fluorescing; (b) determining the phospholipid-induced modulation in florescence of the PH domain, an alteration of the florescence in the presence of the compound being indicative of a functional interaction between the compound and the PH domain.

What is claimed is: 
     
       1. A method for screening for a compound which modulates a signal response caused by a Pleckstin Homology (PH) domain of a signaling molecule comprising the steps of: (a) incubating the compound with the PH domain of a signalling molecule which is capable of fluorescing;   (b) determining the phospholipid-induced modulation in the fluorescence of the PH domain, an alteration of the fluorescence in the presence of the compound being indicative of a functional interaction between the compound and the PH domain.   
     
     
       2. A method according to claim 1 wherein the fluorescence is associated with a Trp residue located in the N-terminus of the PH domain. 
     
     
       3. A method according to claim 1 wherein the signalling molecule is a protein kinase. 
     
     
       4. A method according to claim 3 wherein the signalling molecule is RAC protein kinase and the fluorescent residue is Trp 22 . 
     
     
       5. A method according to claim 1 comprising incubating the candidate modulator compound with purified PH domain. 
     
     
       6. A method according to claim 5 wherein the PH domain is produced by expressing a recombinant expression vector encoding the PH domain as a GST fusion in a bacterial host cell, subsequently cleaving the GST fusion with thrombin and purifying the PH domain. 
     
     
       7. A method according to claim 4 wherein the PH domain is excited at 290 nm and fluorescence is detected at 345 nm.